The failure to properly control inflammatory responses is a common feature in human disease. IL-10 plays a central role in limiting inflammation and IL-10 levels are strongly linked to inflammatory disorders in humans. The levels of IL-10 production are reported to be influenced by single nucleotide polymorphisms (SNPs) in the IL10 promoter and these SNPs are also associated with disease susceptibility. This indicates that inter- individual differences in the regulation of IL-10 production are likely key factor which determines disease risk. However, the mechanisms that control human IL-10 (hIL-10) production remain unclear due to a lack of appropriate research tools. For that reason, we established a proof-of-principle system in the first funding period to transgenically model the regulation of hIL-10 expression in the absence of extraneous genetic and environmental influence. We used a large segment of human genomic DNA flanking the IL10 gene to assure that the regulatory information required to confer appropriate hIL-10 expression would be self-contained (hIL10BAC). In addition, because hIL-10 is functional in mice, we can use this model to establish the connection between hIL-10 regulation and disease outcomes in vivo. We have carefully validated hIL-10 expression in several well-characterized IL-10-dependent disease models as well as in primary human cells. We found that the hIL10BAC rescues Il10-/- mice from LPS toxicity and colitis which was associated with hIL-10 production from macrophages and CD4+FoxP3+ Tregs respectively. Interestingly, the hIL10BAC did not restore susceptibility to persistent L. Donovani infection in Il10-/- mice. This was because; only a small population of hIL-10+Th1 cells (which mediates this phenotype) were induced in hIL10BAC mice. Our findings suggest that hIL-10 is under different cell type-specific regulatory constraints compared to mouse IL-10. Furthermore, because the hIL10BAC contains an IL10 promoter allele associated with low hIL-10 production, these data suggest that IL10 SNPs impart cell-specific hIL-10 expression patterns which result in altered disease outcomes. We now propose to extend these findings to focus on the mechanims which govern cell type- and allele-specific hIL-10 expression patterns as a means to determine the molecular basis for hIL-10's role in human disease outcomes. In Aim 1, we will define how regulatory boundaries and chromatin structure impact hIL-10 expression in different cell types which are implicated in disease pathogenesis and identify the molecular mechanims which control cell-specific hIL-10 expression. For this we have generated new hIL10BAC lines carrying different genomic deletions within the IL10 locus. In Aim 2, we will determine the role of SNPs in hIL-10 production and disease outcomes using new hIL10BAC transgenics carrying an IL10 promoter allele associated with high IL-10 production. The influence of hIL10 SNPs will be validated in primary human cells and the mechanisms of allele-specific hIL-10 expression identified. Together, these studies will clarify how cell- and allele-specific regulation of hIL-10 production contributes to human inflammatory diseases.